The present invention relates, in general, to neurally-differentiated cells infected with viruses in a manner that supports a long-term non-productive infection for experimentation concerning the reactivation, induction, suppressing of virus latency.
Herpes simplex virus types 1 and 2 (HSV-1 and -2) are alphaherpesviruses with similar, but unique molecular (Kieff et. al., 1971, 1972), biological and clinical features (reviewed in Whitley, 1996). The genomes are approximately 150 kb in size and each contains corresponding sets of 74 genes (Dolan et al, 1998). Both viruses infect epithelium, with HSV-1 having a predilection for orofacial sites and HSV-2 preferentially infecting genital surfaces. During the primary infection, HSV invades local nerve endings and travels to sensory ganglia where it can colonize neuronal nuclei and establish a latent state (Hill et al, 1972, Stevens and Cook, 1971). Reactivation of HSV from latency occurs intermittently as a result of stressful stimuli (e.g., trauma and heat). Reactivated viruses are responsible for causing recurrent epithelial infections that can occur in up to 89% of infected individuals (Benedetti, et. al. 1994).
The lack of a universally accepted neural cell culture model that supports HSV latency, in particular, HSV-2, restricts our understanding of the molecular events involved in reactivation from latency. Although animal models reproduce certain aspects of HSV-2 latency in humans (Al-Saadi et al, 1988; Bourne et al, 1994; Croen et al, 1991; Krause et al, 1995; Kurata et al, 1978; MacLean et al, 1991; Martin and Suzuki, 1989; Mitchell et al, 1990; Stanberry et al, 1982; Stephanopoulos et al, 1988; Wang et al, 1997; Yoshikawa et al, 1996), limitations in these models make interpretation of reactivation data challenging. Animal models limitations include: (i) latency and reactivation events that are influenced by viral strains with different primary growth phenotypes, (ii) the limited number of neurons latently infected in animal models (Bloom et al, 1996; Hill et al, 1996; Maggioncalda et al, 1996; Mehta et al, 1995; Ramakrishnan 1994; Sawtell, 1997; Sawtell et al, 1998; Thompson and Sawtell, 1997), and (iii) inaccurate quantitation of reactivation events when measuring virus production at the recurrent site as a result of influences of transport, replication in epithelium, and the immune response.
In response to these limitations, tissue and cell culture models of HSV-2 latency have been developed in an attempt to overcome limitations of animal models. A major advantage of tissue and cell culture models includes the ability to observe virus at the single cell level without the overlay of immunological events that modulate the eventual appearance of virus in the host. In addition, tissue culture models derived from neuronal and sympathetic ganglia have properties of the in vivo system including: (i) restricted transcription of the HSV genome (Doerig et al, 1991; Halford et al, 1996; Smith et al, 1992; Smith et al, 1994), (ii) lack of virus production following removal of the inhibitory agent, (Wilcox and Johnson, 1988) (iii) the presence of latency-associated transcripts (LATs) (Doerig et al, 1991; Smith et al, 1994), (iv) impaired reactivation of thymidine kinase negative virus (Wilcox et al, 1992), and (v) inducible reactivation (Halford et al, 1996; Moriya et al, 1994; Smith et al, 1992; Wilcox and Johnson, 1988; Wilcox and Johnson 1987; Wilcox et al, 1990). Nevertheless, tissue culture models have their drawbacks, preparation of dissected ganglia is inconvenient, material is limited, animal use is required, and axotomy introduces traumatic factors that influence reactivation of virus.
Accordingly, development of cell culture models with neuronal characteristics that lack the restrictive requirements of tissue culture models would be advantageous for understanding the molecular mechanisms of the establishment, maintenance and reactivation stages of HSV latency. Cell culture models also allow for an unlimited supply of a defined host cell and the ability to manipulate genetic material.
Over the past 25 years, cell culture systems using fibroblast cultures (Harris and Preston, 1991; Jamieson et al, 1995; O""Neill, 1977; O""Neill et al, 1972; Russell et al, 1987; Scheck et al, 1989; Wigdahl et al, 1982a; Wigdahl et al, 1982b; Wigdahl et al, 1983) and lymphocytes (Hammer et al, 1981; Youssoufian et al, 1982) have enabled the study of HSV-1 during a latent-like state. These models, however required low input multiplicities and/or the use of replication inhibitors such as anti-viral agents, inhibitory temperatures, or the use of a mutant virus, to prevent virus production. A cell line that has neural morphology and physiology, can survive infection and permit viral production, allow establishment of a long term nonproductive viral infection, and support virus in a state suitable for reactivation studies would be more desirable.
More recently, it has been reported that neurally-differentiated PC12 (ND-PC12) cells can harbor HSV-1 in a quiescent, yet reversible state (Danaher et. al., 1999a). These quiescently infected ND-PC12 cultures (QIF-PC12) demonstrate forskolin- and heat stress (HS)-inducible virus production in a high percentage (50-100%) of cultures for up to 8 weeks after infection, whereas mock-induced cultures maintain the quiescent viral state in the majority of infected cultures (Danaher et al, 1999b). In contrast to these cell culture models, the present invention, however, does not require antiviral conditions to maintain and/or establish the latent-like state (Colberg-Poley et al, 1979, 1981; Harris et al, 1989; Kondo et al, 1990; O""Neill, 1977; O""Neill et al, 1972; Russell et al, 1987; Russell and Preston, 1986; Wigdahl et al, 1981; Wilcox and Johnson, 1988; Wilcox et al, 1990; Yura et al, 1986).
The present application demonstrates that ND-PC12 cells permit establishment of an HSV-2 quiescent state, like HSV-1, following transient acycloguanosine (ACV) treatment. Unlike HSV-1, however, antiviral conditions are not required for the establishment of the HSV-2 quiescent state. In addition, the present invention discloses quiescent cultures in the presence of Vero cells, and the presence of Vero cells enhances the sensitivity to detect HSV-2 produced spontaneously and following induction (i.e., forskolin and HS treatment). Thus, the present invention demonstrates that ND-PC 12 cells can harbor HSV-2, like HSV-1, in a cryptic and non-productive state that is reversible, and this model has appealing features for studying gene induction during the establishment and maintenance of virus latency and the activation of HSV-2 from a nonproductive state.
A primary object of the present application is to provide neurally-differentiated cells infected with viruses in a manner that supports a long-term non-productive infection for experimentation. Another object of the present invention also provides a cell culture research model for HSV-2 quiescent infection in ND-PC12 cells to investigate quiescent and reactivation properties of HSV-2. This model represents an improvement over existing cell culture models for HSV-1. Advantages of this quiescently infected PC12 cell culture model include: (1) establishment and maintenance of a HSV-2 quiescent infection in a high proportion of PC12 cell cultures with and without the transient use of ACV (acyclovir); (2) the ability to produce HSV-2 from a quiescent state in response to forskolin and HS treatment for at lest 4 weeks post infection; and (3) the ability to discriminate between quiescence, spontaneous reactivation and inducible reactivation using a range of multiplicities of infection (MOIs). Thereby, these enhanced features of the invention enable analysis of the establishment and maintenance of latency and the reactivation events of a cryptic HSV genome at the single neural cell level in vitro.
Accordingly, additional objects of the present invention provide for methods of establishing quiescently-infected PC12 cells, reactivating quiescent virus, determining the ability of a test reagent to suppress and/or induce virus reactivation, eliciting phenotypic change in a PC12 cell, determining the susceptibility to reactivation of a person infected with a quiescent virus, identifying nucleic acid molecules and/or proteins involved in virus reactivation, identifying the origins of DNA replication important to virus reactivation, screening an altered virus"" ability to be reactivated, determining the ability of a non-neurotropic virus to become quiescent and/or reactivatable, and a method of determining a reagent""s ability to inhibit establishment of quiescent viral infection.
Additional objects and attendant advantages of the present invention will be set forth, in part, in the description that follows, or may be learned from practicing or using the present invention. The objects and advantages may be realized and attained by means of the instrumentalities and combinations particularly recited in the appended claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be viewed as being restrictive of the invention, as claimed.
The present invention provides, inter alia, neural cells comprising a PC12 cell quiescently infected with a neurotropic virus. Preferred quiescently-infected neural cells are those wherein the neurotropic virus is a neurotropic herpes virus. More preferred are quiescently-infected neural cells wherein the neurotropic virus is a neurotropic human herpes virus. More preferred are quiescently-infected neural cells wherein the neurotropic herpes virus is a human herpes simplex 2 virus. However, those quiescently-infected neural cells wherein the neurotropic virus is selected from the group consisting essentially of varicella zoster viruses, polyoma viruses, measles viruses, human immunodeficiency viruses, papillomaviruses, adenoviruses, cytomegaloviruses, epstein barr viruses, hepatitis viruses, coronaviruses, coxsackie viruses, rabies viruses, flaviviruses, paramyxoviruses, togaviruses, and rhinoviruses, are also preferred.
Also provided are methods of establishing quiescently-infected neural cells, comprising introducing a neurotropic virus to neurally-differentiated and viable PC12 cells in a serum-free medium, said differentiated PC12 cells being in a container; and incubating said container with an antiviral reagent for a time necessary to accomplish quiescence of viral activity; and removing said antiviral agent from said container. Preferred such methods are those wherein the neurotropic virus is a neurotropic herpes virus. More preferred are those methods wherein the neurotropic herpes virus is a human herpes simplex 2 virus. However, those methods wherein the neurotropic virus is selected from the group consisting essentially of varicella zoster viruses, polyoma viruses, measles viruses, human immunodeficiency viruses, papillomaviruses, adenoviruses, cytomegaloviruses, epstein barr viruses, hepatitis viruses, coronaviruses, coxsackie viruses, rabies viruses, flaviviruses, paramyxoviruses, togaviruses, and rhinoviruses are also preferred. Methods wherein the neurotropic herpes virus is a human herpes simplex 2 virus and the antiviral agent is acyclovir are preferred, especially those methods wherein said container is incubated with an antiviral composition for approximately five (5) to twelve (12) days, more especially methods wherein said container is incubated at a temperature less than 40 degrees Celsius (40xc2x0 C.), and most especially wherein said serum free medium allows for constant cell density and imparts neural characteristics to said cells.
Importantly, also provided are methods of establishing quiescently-infected neural cells without using an antiviral reagent, comprising introducing a neurotropic virus to neurally-differentiated and viable PC12 cells in a serum-free medium; replenishing the serum-free medium after introduction of the neurotropic virus to maintain acceptable cell viability. Preferably, the medium is replenished daily for three (3) days, and thereafter, every two (2) to three (3) days until experimentation.
Also provided are methods of reactivating a quiescent virus from neural cell, comprising: introducing a reactivator to any of the herein described quiescently-infected neural cells.
Also provided are methods for determining the ability of a test reagent to suppress virus reactivation from a quiescently-infected neural cell, comprising introducing a test reagent to a quiescently-infected neural cell described herein; and introducing to said neural cell a reactivator; and determining if reactivation has been suppressed.
Also provided are methods for determining the ability of a test reagent to induce virus reactivation in a neural cell, comprising introducing a test reagent to a quiescently-infected neural cell described herein; and determining if reactivation has been induced.
Also provided are methods for determining the ability of a test reagent to establish virus latency in a neural cell, comprising introducing a test reagent to a quiescently-infected neural cell described herein; and determining if latency has been induced.
Also provided are methods for determining the ability of a test reagent to inhibit establishment of a quiescent viral infection, comprising introducing a virus to differentiated and viable PC12 cells; replenishing the serum-free medium after introduction of the non-neurotropic virus to maintain acceptable cell viability. Preferably, the medium is replenished daily for three (3) days, and thereafter, every two (2) to three (3) days until experimentation.
Also provided are methods for eliciting phenotypic change in a neural cell, introducing a reactivator to a quiescently-infected neural cell described herein, and eliciting a phenotypic change in said neural cell. Preferred are such methods wherein said phenotypic change is selected from the group consisting of synthesis of myelin, synthesis of neurotransmitter, cell death, and viral shedding.
Also provided are methods for determining the susceptibility of a person infected with a quiescent virus to reactivation by a reagent, comprising introducing a reactivator to a quiescently-infected neural cell described herein, wherein the neurotropic virus is a strain isolated from a person infected with said neurotropic virus; and determining the relative magnitude of phenotypic or genomic reactivation.
Also provided are methods to identify nucleic acid molecules and/or proteins involved in virus reactivation, comprising reactivating a quiescently-infected neural cell described herein with a reactivator; and identifying nucleic acid molecules and/or proteins which are uniquely expressed during reactivation.
Also provided are methods to identify the origins of DNA replication important to virus reactivation, comprising reactivating a quiescently-infected neural cell described herein with a reactivator; and identifying the origins of replication which are uniquely associated with reactivation.
Also provided are methods to identify nucleic acid molecules and/or proteins involved in establishing and maintaining virus latency, comprising establishing latency of a neural cell described herein according to the methods described herein; and identifying nucleic acid molecules and/or proteins which are uniquely expressed during latency.
Also provided are methods to identify the origins of DNA replication important to establishing and maintaining virus latency, comprising establishing latency of a neural cell described herein according to the methods described herein; and identifying nucleic acid molecules and/or proteins which are uniquely expressed during latency.
Also provided are methods to screen an attenuated virus"" relative ability to be reactivated, comprising introducing a reactivator to a quiescently-infected neural cell described herein, wherein the neurotropic virus is an attenuated virus; and determining the relative magnitude of reactivation.
Also provided are methods to determine the ability of a virus to become quiescent and/or reactivatable in a neural cell line, comprising introducing a non-neurotropic virus to differentiated and viable PC12 cells in a serum-free medium; replenishing the serum-free medium after introduction of the neurotropic virus to maintain acceptable cell viability. Preferably, the medium is replenished daily for three (3) days, and thereafter, every two (2) to three (3) days until experimentation.
For the purposes of the present application, the following terms shall have the following meanings:
xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d, when describing a noun, refers to one or more of that noun.
xe2x80x9cacceptable cell viabilityxe2x80x9d means sufficient viability of quiescently infected cells to perform experiments.
xe2x80x9cantiviral reagentxe2x80x9d means a reagent which prevents viral growth or DNA replication in a cell.
xe2x80x9ccompositionxe2x80x9d means any compound or composition made by any means. xe2x80x9cCompositionxe2x80x9d includes synthetic or naturally-occurring compounds or compositions, whether purified or not, and can include: biologicals, chemicals, herbal extract(s); precursor(s); metabolite(s); and ingredient(s), including enantiomer(s) of a racemic mixture. The definition of xe2x80x9ccompositionxe2x80x9d includes compounds produced in situ by virtue of an immune response (i.e., immunoglobulins and compounds involved in inflammation), as well as organisms, such as: viruses, bacteria and fungi.
xe2x80x9cisolatedxe2x80x9d means physically removed from a form found in nature. For instance, whole cells, a crude cell extract, purified virus, molecularly engineered virus, or artificial virus would be xe2x80x9cisolatedxe2x80x9d virus.
xe2x80x9cneurotropic virusxe2x80x9d means any virus which is capable of infecting neurons, including viruses which only transiently infect neurons.
xe2x80x9cquiescentxe2x80x9d or xe2x80x9cquiescencexe2x80x9d means the absence of detectable infectious particles in the media and within the cells having viral nucleic acid present in the cells.
xe2x80x9creactivatorxe2x80x9d means a reagent which will cause reactivation of quiescent virus.
xe2x80x9creactivationxe2x80x9d means any change in phenotype or genotype from a quiescent state.
xe2x80x9creagentxe2x80x9d means a composition or an environmental condition, temperature, ultraviolet radiation, biological, etc.
xe2x80x9cvirusxe2x80x9d means the definition as understood by those in the art, as well as viroid particles such as prions, and including natural and artificial alterations thereof (eg. mutations (eg. temperature sensitive mutations), including deletions, insertions, etc.)